Supplementary Information
Relationship between the Physicochemical Properties of Lipid Nanoparticles and
the Quality of siRNA Delivery to Liver Cells
Yusuke Sato, Hiroto Hatakeyama, Mamoru Hyodo, and Hideyoshi Harashima
Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences,
Hokkaido University, Kita-12, Nishi-6, Kita-Ku, Sapporo 060-0812, Japan.
General Information
1
H and 13C NMR spectra were measured on a JEOL ECA500, ECX400P or ECS400
instrument, with tetramethylsilane as the internal standard (0 ppm). The following
abbreviations were used to express the peaks: s = singlet; d = doublet; t = triplet; m =
multiplet; br = broad. 1H NMR cheimcal shifts are reported in ppm on the sigma scale
downfield from tetramethylsilane. All reactions were monitored by thin-layer
chromatography on pre-coated TLC plates (Millipore), visualization was achieved by
using UV light (254 nm), phosphomolybdic acid stain or p-anisaldehyde stain. The
products were purified by flash column chromatography on silica gel or an automated
Teledyne ISCO combiflash Rf chromatography system. All ordinary chemicals were
purchased and used without further purification. Compound 1 was prepared via a
previously reported method.1
Methods
Synthesis of pH-sensitive cationic lipids.
Synthesis of tert-butyl (12Z,15Z)-3-((9Z,12Z)-octadeca-9,12-dien-1-yl)henicosa2,12,15-trienoate (2). tert-Butyl diethylphosphonoacetate (2.14 mL, 9.11 mmol) and tBuOK (937 mg, 8.35 mmol) were successively added to anhydrous t-BuOH (40 mL) and
the resulting solution stirred at room temperature for 5 min, and the ketone 1 (4.00 g, 7.59
mmol) dissolved in anhydrous t-BuOH (10 mL) was then added. The mixture was
refluxed for 3 h and then concentrated. The residue was dissolved in EtOAc (50 mL), and
the organic layer was washed with a saturated NH4Cl solution (2×50 mL). The organic
phase was dried over anhydrous Na2SO4. Evaporation of the solvent resulted in a
yellowish oily residue. The residue was chromatographed on silica gel (SiO2,
hexane/EtOAc) to give 3.54 g (75%) of 2 as a colorless oil. 1H NMR (500 MHz, CDCl3)
: 5.52 (1H, s), 5.32-5.37 (8H, m), 2.76 (4H, t), 2.53 (2H, t), 1.97-2.09 (10H, m), 1.47
(9H, s), 1.20-1.45 (36H, m), 0.87 (6H, t). 13C NMR (101 MHz, CDCl3) : 163.31, 162.78,
130.26, 128.01, 117.02, 79.41, 77.43, 77.10, 76.78, 38.40, 31.90, 31.62, 30.01, 29.86,
29.78, 29.61, 29.54, 29.44, 28.77, 28.36, 27.84, 28.29, 25.71, 22.67, 14.17. HRMS (m/z)
Calcd. for C43H76O2Na (M+Na)+ 647.5738; Found 647.5735.
Synthesis
of
(12Z,15Z)-3-((9Z,12Z)-octadeca-9,12-dien-1-yl)henicosa-2,12,15trienoic acid (3). tert-Butyl ester 2 (453 mg, 0.725 mmol) was dissolved in
dichloromethane (DCM), and trifluoroacetic acid (TFA, 10%) was added. The mixture
was stirred at room temperature for 4 h. EtOAc (50 mL) was added to the mixture, which
was then washed with a NaOH solution (1 M, 2×50 mL), a HCl solution (1 M, 50 mL)
and brine (50 mL). The organic phase was dried over Na2SO4. Evaporation of the solvent
gave crude 3 as a pale yellow oily residue, which was used in the following step without
further purification.
Synthesis of 2-(dimethylamono)ethyl (12Z,15Z)-3-((9Z,12Z)-octadeca-9,12-dien-1yl)henicosa-2,12,15-trienoate (YSK13-C2). Acid 3 (569 mg, 1.00 mmol) was dissolved
in DCM (20 mL) and DMAP (122 mg, 1.00 mmol) and EDCI (959 mg, 5.00 mmol) were
added to this mixture. After stirring for 1 h at ambient temperature, dimethylaminoethanol
(503 L, 5.00 mmol) was added and the reaction mixture was stirred at ambient
temperature overnight. The reaction mixture was diluted with DCM (50 mL) and washed
with a NaOH solution (1 M, 2×50 mL) and brine (50 mL). The organic phase was dried
over Na2SO4 and the solvents were removed in vacuo. The crude product was
chromatographed on silica gel (SiO2, DCM/MeOH) to give 539 mg of YSK13-C2 as a
pale yellow oil. 1H NMR (500 MHz, CDCl3) : 5.66 (1H, s), 5.29-5.41 (8H, m), 4.17 (2H,
t), 2.77 (4H, t), 2.58 (2H, t), 2.29 (6H, s), 1.97-2.13 (12H, m), 1.20-1.47 (36H, m), 0.88
(6H, t). 13C NMR (101 MHz, CDCl3) : 166.62, 165.65, 130.27, 128.01, 114.86, 77.44,
77.12, 76.80, 61.25, 58.05, 45.82, 45.82, 38.52, 32.28, 31.62, 30.09, 29.78, 29.62, 29.54,
29.44, 28.77, 27.74, 27.29, 25.71, 22.67, 14.18. HRMS (m/z) Calcd. for C43H78NO2
(M+H)+ 640.6027; Found 640.6015.
Synthesis of 2-(dimethylamono)propyl (12Z,15Z)-3-((9Z,12Z)-octadeca-9,12-dien-1yl)henicosa-2,12,15-trienoate (YSK13-C3). Acid 3 (3.00 g, 5.28 mmol) was dissolved
in DCM (26 mL) and DMAP (323 mg, 2.64 mmol) and EDCI (2.53 g, 13.2 mmol) were
added. After stirring for 1 h at ambient temperature, dimethylaminopropanol (1.54 mL,
13.2 mmol) was added and the reaction mixture was stirred at ambient temperature
overnight. The reaction mixture was diluted with DCM (100 mL) and washed with a
NaOH solution (1 M, 2×100 mL) and brine (100 mL). The organic phase was dried over
Na2SO4 and the solvents were removed in vacuo. The crude product was
chromatographed on silica gel (SiO2, DCM/MeOH) to give 1.90 g of YSK13-C3 as a
pale yellow oil. 1H NMR (400 MHz, CDCl3) : 5.60 (1H, s), 5.29-5.40 (8H, m), 4.10 (2H,
t), 2.76 (4H, t), 2.57 (2H, t), 2.33 (2H, t), 2.21 (6H, s), 2.12 (2H, t), 1.97-2.09 (8H, m),
1.81 (2H, m), 1.20-1.45 (36H, m), 0.88 (6H, t). 13C NMR (101 MHz, CDCl3) : 166.66,
165.21, 130.27, 128.01, 114.96, 77.43, 77.11, 76.79, 61.99, 56.50, 45.60, 38.52, 32.26,
31.62, 30.09, 29.86, 29.78, 29.62, 29.55, 29.44, 28.82, 27.78, 27.29, 27.24, 25.71, 22.67,
14.18. HRMS (m/z) Calcd. for C44H80NO2 (M+H)+ 654.6184; Found 654.6173.
Synthesis of 2-(dimethylamono)butyl (12Z,15Z)-3-((9Z,12Z)-octadeca-9,12-dien-1yl)henicosa-2,12,15-trienoate (YSK13-C4). Acid 3 (285 mg, 0.50 mmol) was dissolved
in DCM (10 mL) and to it DMAP (61.1 mg, 0.50 mmol) and EDCI (479 mg, 2.5 mmol)
were added. After stirring for 1 h at ambient temperature, dimethylaminopropanol (333
L, 2.5 mmol) was added and the reaction mixture was stirred at ambient temperature
overnight. The reaction mixture was diluted with DCM (50 mL) and washed with a NaOH
solution (1 M, 2×50 mL) and brine (50 mL). The organic phase was dried over Na2SO4
and the solvents were removed in vacuo. The crude product was chromatographed on
silica gel (SiO2, DCM/MeOH) to give 228 mg of YSK13-C4 as a pale yellow oil. 1H
NMR (400 MHz, CDCl3) : 5.59 (1H, s), 5.27-5.40 (8H, m), 4.08 (2H, t), 2.76 (4H, t),
2.56 (2H, t), 2.28 (2H, t), 2.21 (6H, s), 2.12 (2H, t), 1.97-2.09 (8H, m), 1.65 (2H, m),
1.20-1.55 (38H, m), 0.88 (6H, t). 13C NMR (101 MHz, CDCl3) : 166.73, 165.18, 130.27,
128.01, 114.99, 77.44, 77.12, 76.80, 63.45, 59.40, 63.45, 59.40, 45.54, 38.52, 31.62,
29.78, 29.61, 29.54, 29.44, 27.29, 25.71, 22.67, 14.18. HRMS (m/z) Calcd. for C45H82NO2
(M+H)+ 668.6340; Found 668.6321.
Synthesis of (12Z,15Z)-3-((9Z,12Z)-octadeca-9,12-dien-1-yl)henicosa-2,12,15-trien1-ol (4). Aluminum chloride (581 mg, 4.36 mmol) in dry ether (15 mL) was added
dropwise to a stirred, ice-cooled suspension of lithium aluminium hydride (248 mg, 6.54
mmol) in anhydrous ether (15 mL). After stirring the hydride mixture at ice-bath
temperature for 30 min, the tert-butyl ester 2 (1.36 g, 2.18 mmol) in anhydrous ether (10
mL) was added dropwise. The reaction mixture was stirred for an additional 1 h at icebath temperature. The reaction mixture was quenched with ice-cold water and then
filtered through a celite pad. Evaporation of solvent gave the crude reduced alcohol. The
crude product was chromatographed on silica gel (SiO2, hexane/EtOAc) to give 689 mg
of 4 as a colorless oil. 1H NMR (500 MHz, CDCl3) : 5.27-5.41 (9H, m), 4.12 (2H, d),
2.76 (4H, t), 1.94-2.09 (12H, m), 1.20-1.45 (36H, m), 0.88 (6H, t). 13C NMR (101 MHz,
CDCl3) : 144.66, 130.28, 128.05, 123.27, 77.44, 77.12, 76.80, 59.32, 36.88, 31.63, 30.47,
29.80, 29.77, 29.61, 29.57, 29.45, 29.41, 28.99, 28.08, 27.30, 25.71, 22.67, 14.18. HRMS
(m/z) Calcd. for C39H70ONa (M+Na)+ 577.5319; Found 577.5313.
Synthesis of (12Z,15Z)-3-((9Z,12Z)-octadeca-9,12-dien-1-yl)henicosa-2,12,15-trien1-yl 3-(dimethylamino)propanoate (YSK15-C2). Alcohol 4 (331 mg, 0.60 mmol) was
dissolved in DCM (5 mL) and DMAP (29.1 mg, 0.24 mmol), triethylamine (TEA) (249
L, 1.79 mmol) and 3-N,N-dimethylamino propanoic acid hydrochloride (220 mg, 1.43
mmol) were added to the solution. After stirring for 5 min at ambient temperature, EDCI
(343 mg, 1.79 mmol) was added and the reaction mixture was stirred at ambient
temperature for 4 h. The reaction mixture was siluted with DCM (50 mL) and washed
with NaOH solution (1 M, 2×50 mL) and brine (50 mL). The organic phase was dried
over Na2SO4 and the solvents were removed in vacuo. The crude product was purified
with fluch column chromatography (SiO2, DCM/MeOH). This gave 376 mg of YSK15C2 as a colorless oil. 1H NMR (500 MHz, CDCl3) : 5.28-5.41 (9H, m), 4.59 (2H, d),
2.77 (4H, t), 2.61 (2H, t), 2.47 (2H, t), 2.23 (6H, s), 1.94-2.07 (12H, m), 1.20-1.42 (36H,
m), 0.88 (6H, t). 13C NMR (101 MHz, CDCl3) : 172.63, 147.12, 130.27, 128.04, 118.04,
77.44, 77.12, 76.80, 61.35, 54.86, 45.39, 31.62, 29.77, 29.62, 29.44, 29.41, 27.33, 27.29,
25.71, 22.67, 14.18. HRMS (m/z) Calcd. for C44H80NO2 (M+H)+ 654.6184; Found
654.6176.
Synthesis of (12Z,15Z)-3-((9Z,12Z)-octadeca-9,12-dien-1-yl)henicosa-2,12,15-trien1-yl 3-(dimethylamino)butanoate (YSK15-C3). Alcohol 4 (277.5 mg, 0.50 mmol) was
dissolved in DCM (4 mL) and DMAP (24.4 mg, 0.20 mmol), TEA (209 L, 1.50 mmol)
and 4-(dimethylamino)butyric acid hydrochloride (201 mg, 1.20 mmol) were then added.
After stirring for 5 min at ambient temperature, EDCI (288 mg, 1.50 mmol) was added
and the reaction mixture was stirred at ambient temperature overnight. The reaction
mixture was diluted with DCM (50 mL) and washed with a NaOH solution (1 M, 2×50
mL) and brine (50 mL). The organic phase was dried over Na2SO4 and solvents were
removed in vacuo. The crude product was chromatographed on silica gel (SiO2,
DCM/MeOH) to give 305 mg of YSK15-C3 as a colorless oil. 1H NMR (500 MHz,
CDCl3) : 5.28-5.41 (9H, m), 4.58 (2H, d), 2.76 (4H, t), 2.32 (2H, t), 2.28 (2H, t), 2.20
(6H, s), 1.95-2.08 (12H, m), 1.78 (2H, m), 1.20-1.40 (36H, m), 0.88 (6H, t). 13C NMR
(101 MHz, CDCl3) : 173.72, 146.96, 130.27, 128.04, 118.15, 77.43, 77.11, 76.79, 61.21,
58.95, 45.49, 36.9, 32.35, 31.62, 30.68, 29.77, 29.62, 29.54, 29.44, 29.41, 28.87, 27.95,
27.33, 27.29, 25.71, 23.09, 22.67, 14.18. HRMS (m/z) Calcd. for C45H82NO2 (M+H)+
668.6340; Found 668.6332.
Synthesis of (12Z,15Z)-3-((9Z,12Z)-octadeca-9,12-dien-1-yl)henicosa-2,12,15-trien1-yl 3-(dimethylamino)pentanoate (YSK15-C4). Alcohol 4 (201 mg, 0.36 mmol) was
dissolved in DCM (4 mL) and to it DMAP (17.7 mg, 0.15 mmol), TEA (252 L, 1.81
mmol) and 5-(dimethylamino)pentanoic acid (90 mg, 0.63 mmol) were added. After
stirring for 5 min at ambient temperature, EDCI (347 mg, 1.81 mmol) was added and the
reaction mixture was stirred at ambient temperature overnight. The reaction mixture was
siluted with DCM (50 mL) and washed with a NaOH solution (1 M, 2×50 mL) and brine
(50 mL). The organic phase was dried over Na2SO4 and the solvents were removed in
vacuo. The crude product was chromatographed on silica gel (SiO2, DCM/MeOH) to give
211 mg of YSK15-C4 as a pale yellow oil. 1H NMR (500 MHz, CDCl3) : 5.28-5.41 (9H,
m), 4.58 (2H, d), 2.76 (4H, t), 2.32 (2H, t), 2.28 (2H, t), 2.20 (6H, s), 1.95-2.10 (12H, m),
1.64 (2H, m), 1.49 (2H, m), 1.20-1.40 (36H, m), 0.88 (6H, t). 13C NMR (101 MHz,
CDCl3) : 173.70, 146.99, 130.27, 128.04, 118.13, 77.43, 77.11, 76.79, 61.17, 59.38,
45.50, 36.91, 34.31, 31.62, 30.68, 29.77, 29.61, 29.44, 27.40, 28.87, 27.95, 27.32, 27.29,
27.20, 25.71, 22.94, 22.67, 14.18. HRMS (m/z) Calcd. for C46H84NO2 (M+H)+ 682.6497;
Found 682.6488.
Synthesis of (12Z,15Z)-3-((9Z,12Z)-octadeca-9,12-dien-1-yl)henicosa-2,12,15-trien1-yl 3-(dimethylamino)hexanoate (YSK15-C5). Alcohol 4 (201 mg, 0.36 mmol) was
dissolved in DCM (8 mL) and to it DMAP (17.7 mg, 0.15 mmol), TEA (252 L, 1.81
mmol) and 6-(dimethylamino)hexanoic acid (94 mg, 0.66 mmol) were added. After
stirring for 5 min at ambient temperature, EDCI (347 mg, 1.81 mmol) was added and the
reaction mixture was stirred at ambient temperature overnight. The reaction mixture was
diluted with DCM (50 mL) and washed with a NaOH solution (1 M, 2×50 mL) and brine
(50 mL). The organic phase was dried over Na2SO4 and the solvents were removed in
vacuo. The crude product was chromatographed on silica gel (SiO2, DCM/MeOH) to give
173 mg of YSK15-C5 as a pale yellow oil. 1H NMR (500 MHz, CDCl3) : 5.28-5.41 (9H,
m), 4.58 (2H, d), 2.35-2.57 (6H, br), 2.76 (4H, t), 2.31 (2H, t), 1.93-2.09 (12H, m), 1.63
(4H, m), 1.20-1.40 (40H, m), 0.88 (6H, t). 13C NMR (101 MHz, CDCl3) : 173.83, 147.00,
130.27, 128.04, 118.14, 77.44, 77.12, 76.80, 61.15, 59.61, 45.42, 36.91, 34.38, 31.62,
30.68, 29.77, 29.61, 29.55, 29.44, 29.40, 28.86, 27.95, 27.32, 27.29, 27.07, 25.71, 25.00,
22.67, 14.17. HRMS (m/z) Calcd. for C47H87NO2 (M+H)+ 696.6653; Found 696.6646.
Table S1. Physiological parameters of MENDs.
Data are represented as the mean±SD (n = 3) or the mean (n = 2).
Table S2. List of siRNA sequences used in this study.
2’-OMe modified nucleotides are in lower case letters, 2’-fluoro modified nucleotides are in bold
lower case letters, and phosphorothioate linkages are represented by asterisks.
Table S3. List of PCR primers used in this study.
Figure S1. Intrahepatic localization of siRNAs formulated in 3 kinds of YSK15MENDs (pKa 6.65 - 7.25). Mice were intravenously injected with MENDs encapsulating
Cy5-labeled siRNAs. Liver tissues were collected, and blood vessels were stained with
FITC-conjugated Isolectin B4. Blood vessels and siRNAs are visualized as green and red,
respectively. Bars represent 50 m.
Figure S2. ApoE-dependency of gene silenicing in hepatocytes and LSECs. (a) ApoEdeficient mice were injected with YSK13-C3-MEND at 0.1 mg kg-1. (b) ApoE-deficient
mice were injected with YSK05-MEND or YSK13-C3-MEND at 0.3 mg kg-1. ApoE was
added to the MEND solution prior to injection and the dose of ApoE was fixed at 0.1 mg
kg-1. The data are represented as the mean±SD (n = 3). *P < 0.05, **P < 0.01 (by oneway nrANOVA, followed by SNK test).
Figure S3. Efficient and liver specific CD31 gene silencing of YSK15-C4-MEND. (a)
Dose-dependency of hepatic CD31 gene silencing. (b) CD31 expression in lung, heart,
kidneys and spleen after the injection of YSK15-C4-MEND at 0.3 mg kg-1. siRNA against
polo-like kinase 1 (siPLK1) was used as a control siRNA. The data are represented as the
mean±SD (n = 3).
Figure S4. Effect of GSK264220A on FVII gene silencing activity in liver. Mice were
intraperitoneally injected with GSK264220A (30 mg kg-1) or vehicle and intravenously
injected with siFVII formulated in the YSK05-MEND at 0.1 mg kg-1 or the YSK13-C3MEND at 0.01 mg kg-1. Plasma FVII activity was measured in 24 hours. Data are
represented as the mean±SD (n = 3).
References
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J., Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in
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